While it is clear that HIV-1 assembly occurs predominantly on the plasma membrane (PM), the itinerary that the HIV-1 Gag precursor follows to reach this destination remains ill defined. Likewise, the host cell machinery that promotes Gag trafficking to the PM is incompletely understood. We and others have demonstrated that the matrix (MA) domain of Gag regulates targeting to the site of virus assembly, and we discovered that the phosphoinositide PI(4,5)P2 is a key player in directing Gag to the PM. We hypothesize that host factors in addition to PI(4,5)P2 play a vital role in the trafficking of Gag to the PM. In this project, we are defining the role of host cell machinery in Gag trafficking to the PM and in subsequent steps of particle assembly and release. This effort combines virology, biochemistry, cell biology, and imaging techniques. Our overarching goal is to establish a unified view of the mechanism and pathway(s) involved in the late stages of the HIV-1 replication cycle and to develop inhibitors of HIV-1 Gag trafficking, assembly, and release._____ Most of our efforts, and those of our colleagues, with regard to late-acting host factors have focused on factors that promote HIV-1 particle assembly and release. However, we have become increasingly interested in cellular factors that interfere with the late stages of HIV-1 replication. The best-characterized example of a late-acting negative factor is tetherin (BST-2), an interferon-stimulated gene product that restricts viral particle release at the cell surface. Tetherin is counteracted by the HIV-1 Vpu protein and the Nef or Env proteins of certain strains of HIV-2 and simian immunodeficiency virus. Apart from tetherin, however, little is known about the host cell factors that interfere with HIV-1 assembly or release. We are engaged in studying these host proteins, with an initial focus on the T-cell immunoglobulin and mucin domain (TIM) family of phosphatidylserine-binding proteins and ISG15, one of the most strongly upregulated proteins following type 1 interferon stimulation. We recently reported, in collaboration with Dr. Shan-Lu Liu's lab (University of Missouri), that the TIM-family proteins strongly inhibit HIV-1 release by retaining HIV-1 particles on the cell surface through binding to phosphatidylserine on the viral membrane. We will further characterize the mechanism of action by which such host factors restrict HIV-1 particle production, resulting in dimished viral production and replication._____[Corresponds to Freed Project 1 in the July 2016 site visit report of the HIV Dynamics and Replication Program]